Phosphoenolpyruvate carboxylase is an enzyme which is found in almost all bacteria and all plants. The role of this enzyme resides in biosynthesis of aspartic acid and glutamic acid, and supply of C4 dicarboxylic acid to the citric acid cycle for maintaining its turnover. However, in the fermentative production of an amino acid using a microorganisms, there have been few reports on effects of this enzyme has not been made clear (Atsushi Yokota and Isamu Shiio, Agric. Biol. Chem., 52, 455-463 (1988), Josef Cremer et al., Appl. Environ. Microbiol., 57, 1746-1752 (1991), Petra, G. Peters-Weintisch, FEMS Microbiol. Letters, 112, 269-274 (1993)).
By the way, the amino acid is a compound which universally exists in cells as components of proteins, however, for the sake of economic energy metabolism and substance metabolism, its production is strictly controlled. This control is principally feedback control, in which the final product of a metabolic pathway inhibits the activity of an enzyme which catalyzes the earlier step of the pathway. Phosphoenolpyruvate carboxylase also undergoes various regulations in expression of its activity.
For example, in the case of phosphoenolpyruvate carboxylase of microorganisms belonging to the genus Corynebacterium or the genus Escherichia, the activity is inhibited by aspartic acid. Therefore, the aforementioned amino acid biosynthesis, in which phosphoenolpyruvate carboxylase participates, is also inhibited by aspartic acid.
In the prior art, various techniques have been developed for efficient production in amino acid fermentation, and fermentative production has been carried out for leucine, isoleucine, tryptophan, phenylalanine and the like by using mutant strains converted to be insensitive to feedback control. However, there has been known neither mutant phosphoenolpyruvate carboxylase converted to be insensitive to inhibition by aspartic acid, nor attempt to utilize it for fermentative production of amino acids of the aspartic acid family and the glutamic acid family.
On the other hand, ppc gene, which is a gene coding for phosphoenolpyruvate carboxylase of Escherichia coli, has been already cloned, and also determined for its nucleotide sequence (Fujita, N., Miwa, T., Ishijima, S., Izui, K. and Katsuki, H., J. Biochem., 95, 909-916 (1984)). However, there is no report of a mutant in which inhibition by aspartic acid is desensitized.
The present invention has been made from the aforementioned viewpoint, an object of which is to provide a mutant phosphoenolpyruvate carboxylase with substantially desensitized feedback inhibition by aspartic acid, a gene conding for it, and a utilization method thereof.